This work was supported by NIH grant 1R01EY022901, the Career Development Award from Research to Prevent Blindness (RPB), C.M.Reeves &#38; M.A. Reeves Foundation, E. Matilda Ziegler Foundation for the Blind, Knights Templar Eye Foundation, and an unrestricted grant to the Department of Ophthalmology at the University of Utah from RPB. &#160;We thank Balamurali Ambati for technical assistance on the Spectralis Multi-Modality Imaging System and Tao Zhang for discussions and comments on the manuscript.

1. Preparation of Instruments
<ol>
	<li>The procedure is performed in a procedure room in an animal facility.</li>
	<li>Wear facemasks, hair bonnets, surgical gowns, sterile foot-covers, and gloves before beginning the experiment.</li>
	<li>Heat water in a beaker to ~40 &#176;C on a hotplate.</li>
	<li>Place a sterile blue pad on top of a heating pad that will be used later to maintain the mouse&#39;s body temperature during imaging. Turn on the heating pad.</li>
	<li>Prepare the Imaging System:
	<ol>
		<li>Remove the dust cover and turn on the laser.</li>
		<li>Take out the 55&#176; lens and mount it onto the machine.</li>
		<li>Open the imaging software from the computer and input the information of the mouse for imaging under a new patient&#39;s sheet (e.g. genotype, age, etc.). Under &#34;device type&#34;, choose Infrared (IR) mode.</li>
	</ol>
	</li>
</ol>
Note: It has been reported that the use of an external double aspheric lens can improve the image quality17-20 although we have no problem in obtaining high quality images by IV-ICGA without using external lenses (see representative results, Figures 1-4).
2. Tail Vein Injection of ICGA
<ol>
	<li>Dilate mouse eyes with 1% Tropicamide ophthalmic solution and wait 5 min.</li>
	<li>Weigh the mouse to determine the amount of anesthesia (Ketamine/Xylazine/Acepromazine 65-100/10-20/1-3 mg/kg) needed.</li>
	<li>Retrieve a sterile 1-ml&#160;syringe along with a 32&#160;G&#160;needle. Inject the mouse intraperitoneally with the anesthetics (13 mg/ml&#160;Ketamine, 2.6 mg/ml Xylazine, 0.3 mg/ml&#160;Acepromazine in sterile PBS). Wait until the mouse is fully anesthetized (~5 min).</li>
	<li>Place the mouse tail into 40 &#176;C warm water to cause vasodilatation of the vein.</li>
	<li>Retrieve a 1&#160;ml&#160;syringe with a 32&#160;G&#160;needle. Withdraw the desired amount of ICG, typically 50 &#956;l&#160;of 1 mg/ml&#160;ICG, which is sterile filtered with a 0.2 &#181;M syringe filter into a sterile tube,&#160;for a 25 g mouse (2 mg/kg). Be careful not to introduce any air into the syringe.</li>
	<li>Wipe the tail with an alcohol swab to sterilize the area to be injected.</li>
	<li>Hold the tail with one hand so that the lateral tail vein is upward. With the bevel of the needle facing upward, inject the needle ~2 mm into the vein at a minimal angle. Be careful not to perforate the vein. Draw back on the syringe slightly and look for traces of blood flow into the needle hub, which indicates that the needle was successfully inserted into the vein.</li>
	<li>Slowly inject ICG into the vein. There should be minimal resistance when injecting. Remove the needle and apply an alcohol swab directly to the injection site for ~5-10 sec&#160;to stop any bleeding. The mouse is then ready for imaging. In order to catch the early phase (0-4 min after injection), it is essential to image the mouse quickly.</li>
</ol>
Note: Mouse eyes can easily get dry and can develop cataract under anesthesia. It is important to keep the eye moist by applying sterile PBS during the procedure. Wipe off excess PBS with a sterile cotton swab before ICGA recording. Other labs have used a contact lens to avoid dehydration of the cornea17-20.
3. ICG Angiography
<ol>
	<li>Start to take images 30-40 sec&#160;after ICG injection, which permits the capture of the early phase of choroidal filling until retinal and choroidal circulations are at maximum brightness (0-4 min). The retinal vasculature is best visualized at focus ~35-45 diopters and choroidal vasculature is visualized at 10-15 diopters. 
	 
	Note: During the first examination of an animal model, it is recommended to capture images from all angles (nasal, temporal, dorsal and ventral) to identify all possible abnormalities in the vasculature. During the early phase, both medium and large choroidal arteries and veins are well visualized. In the animal model used in this protocol, choroidal lesions (e.g. polypoidal dilations) may start to appear 1 min into the early phase.</li>
	<li>Set the image focus on the vasculature. Control for brightness and focus using the control module and focus knob, respectively. These values are adjustable digitally and are easily kept constant. Keep the distance from the mouse eye to the camera lens constant to ensure image quality is reproducible using the technique that follows. 
	 
	Note: Since the device can only image a portion of the posterior eye, we try to keep the focus, brightness, and the distance between the camera lens and the mouse eye constant as we image the entire posterior eye from different angles. The key to this is to align the circle-shaped luminescence emitted by the ICG through the eye with the field of view of the camera. This is accomplished by making left-to-right, up-and-down, and in-and-out adjustments of the camera position until the entire image has no dark areas. When the luminescence and the field of view of the camera are lined up, the distance from the eye to the lens will be reproducible for the next set of images as well as at an optimal distance for quality imaging.</li>
	<li>Once the vasculature is in focus, capture the image frames by pressing the round black button on the acquisition module. The round black button can also be used to reduce or enhance the signal of ICG for best image quality.</li>
	<li>Determine the optimal view angle and focusing depth to image choroidal lesions. It is important to keep the position of the eye, the focusing depth, and other device settings fixed for the entire time-course ICGA. The images are saved by pressing acquire button on the touch screen panel of the acquisition module.</li>
	<li>Acquire images in the middle phase at 6-15 min after injection. 
	 
	Note: Both the choroidal and retinal vessels become less distinct. Choroidal vasculature appears as diffuse fluorescence. Choroidal lesions exhibiting hyperfluorescence emerge in contrast to the fading surrounding normal background fluorescence.</li>
	<li>Acquire images in the late phase at 17-25 min after injection. 
	 
	Note: Hyperfluorescence fades. Both choroidal and retinal vessels are no longer visible. The optic nerve head becomes black. Hyperfluorescent choroidal lesions have maximal contrast with the fading background.</li>
	<li>After finishing the acquisition of images, apply a clear lubricant eye gel to mouse eyes and leave the mouse on a heating pad for recovery.</li>
	<li>Return mice to their cages and holding area. Export images as Tiff or JPEG files for further analysis.</li>
</ol>
Note: The timing of each phase is not absolute. We found that the timing of each phase might change depending on the amount of ICG injected. More ICG tends to prolong each phase. The best way to define a phase is according to the key features of each phase listed above.

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YF is an inventor of two pending patents that are relevant to the AMD mouse model used in this work.&#160;SK, ZB, and ADJ have nothing to disclose.

In this study, we demonstrated the use of ICGA to image choroidal lesions in HTRA1 transgenic mice. The characteristics of the early, middle, and late phases of ICGA in our mouse model match the time course well in human studies1. This is important to make better comparisons between human pathology and animal phenotypes, which are invaluable for research on pathophysiological mechanisms and treatment strategies of conditions related to the choroid such as AMD.
We first performed ICGA in mice by IP injection and found that the time course was highly variable from mouse to mouse, probably due to the variable absorption of the ICG dye from the body cavity in the abdomen (Figure 3). This makes it difficult to compare with human studies performed by IV injection. In addition, the angiographic features of different phases of IV-ICGA are very useful for characterizing different types of choroidal lesions in animal models. Most people choose to avoid IV injection (e.g. in FA) for mice due to the technical challenge of performing tail vein injection (mouse tail veins are tiny). However, the effort is well spent considering the reproducible nature of this technique and the amount of information obtained. Once we mastered the technique of tail vein injection, the other steps are rather similar to IP-ICGA. It is worthwhile to mention that one has to get everything prepared beforehand (e.g. the Imaging System) in order to capture the very short early phase (0-4 min). We compared IP-ICGA vs. IV-ICGA for studying various HTRA1 transgenic mice. We have done ~100 mice for each method. The conclusion is that IV-ICGA is superior to IP-ICGA for characterizing lesions in the choroid. Time-course IV-ICGA has become our standard practice to examine AMD mouse models. For the same reason, we suggest that researchers should consider performing IV-FA for animal research.
Other than the injection route, we noticed that pigment color also influences the quality of ICGA. Previous studies also reported this &#34;pigmentation effect&#34;18,25. However, no information is available on the influence of the coat color on the different phases of ICGA. We compared ICGA between pigmented mice (C57Bl6) and albino mice (CD1) by time course IV-ICGA. Big choroidal vessels appear fuzzy and less clear while the small vessels are difficult to see in C57Bl6 mice, which is in sharp contrast to the much sharper images of both big and small choroidal vessels in CD1 mice (Figure 4). The biggest difference was observed in the early phase although the middle phase is also affected. There is no big difference in the late phase ICGA due to the fading of the ICG signal in the choroidal vessels. Apparently, ICG fluorescence can be partially blocked by the RPE and melanocytes in the choroid in pigmented mice. As a suggestion, one may want to consider breeding their AMD models into the CD1 background to obtain high resolution ICGA.
Although FA is more widely used on AMD animal models, ICGA is essential in detecting abnormalities in the choroidal vasculature. The ability to observe mouse choroidal vasculature at high resolution in real time can greatly help researchers in characterizing AMD mouse models and in correlating with histopathological data. The combination of ICGA, FA and OCT will be extremely useful in characterizing the phenotype of AMD models as in the diagnosis of AMD in human patients. Since the mouse is currently the most widely used animal model for AMD research26-29, time-course IV-ICGA can play a wider role in the research community.

Indocyanine green angiography (ICGA) is a diagnostic test to image problems related to blood vessels in the eye. The absorption spectrum of ICG ranges from 790-805 nm while the emission spectrum ranges from 770-880 nm with the peak emission at 835 nm&#160;1. This is different from the other popular dye, sodium fluorescein, whose spectrum falls in the visible range. The infrared spectrum enables ICG to penetrate through retinal pigment epithelium (RPE), serosanguineous fluid, and lipid exudates, all of which can easily block visualization by sodium-fluorescein based fluorescein angiography (FA). ICG is 98% protein-bound in the vasculature resulting in less extravasation, allowing enhanced imaging of choroidal vessels and choroidal lesions1,2. ICGA is almost the only choice to visualize choroidal vasculature, which is posterior to RPE. Figure 1 shows the comparison of ICGA and FA in imaging vasculature in mouse eyes. FA can be used to image the retinal vasculature well but not the choroidal vasculature. In contrast, ICGA can be used to image both retinal and choroidal vasculature. ICGA is performed with high-resolution digital imaging systems or scanning laser ophthalmoscopes (SLO) together with infrared-sensitive video cameras, which we will use in this study.
In the clinic, ICGA has been recommended in diagnosing a number of chorioretinal disorders involving the choroidal vasculature including Polypoidal Choroidal Vasculopathy (PCV), Retinal Angiomatous Proliferation (RAP), angioid streaks, vitelliform macular dystrophy, central serous chorioretinopathy, choroidal hemangioma, hemorrhaging retinal arteriolar macroaneurysms, choroidal tumors, and certain forms of posterior uveitis1,3. The combination of ICGA with FA and Optical Coherence Tomography (OCT) provide powerful tools for the clinicians in the diagnosis and management of exudative age-related macular degeneration (AMD)4-10. ICGA is especially useful for diagnosing conditions involving the choroid. In fact, ICGA is considered the gold standard for diagnosing PCV, a variant of exudative AMD11-13. PCV is characterized by a network of branching vessels with terminal polypoidal dilations in the choroidal vasculature11-13. PCV is frequently associated with recurrent serosanguineous detachments of the RPE and retina with leakage and bleeding from the polypoidal components11,14,15. We recently reported the generation of the first PCV animal model by transgenically expressing human HTRA1, a multi-functional serine protease, in mouse retinal pigment epithelium (RPE)16. We showed that increased HTRA1 induced characteristic features of PCV, e.g. polypoidal lesions.
Here we demonstrated the use of time-course ICGA by tail vein injection in AMD research using our HTRA1 mouse model. Our data suggest that IV-ICGA is superior to IP (or subcutaneous (SC))-ICGA that are currently used in the field17,18 for characterizing lesions in the choroid.
Statement on Animal Research&#160;
Animal experiments were conducted according to protocols approved by Institutional Animal Care and Use Committee (IACUC), and were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

<table><tbody><tr><td>Spectralis Multi-Modality Imaging System</td><td>Heidelberg Engineering, Germany</td><td>SPECTRALIS HRA+OCT</td><td></td></tr><tr><td>Tropicamide ophthalmic solution (1%)</td><td>Bausch &amp;amp; Lomb</td><td>NDC 24208-585-64</td><td>for dilation of pupils</td></tr><tr><td>GenTeal Gel</td><td>Genteal</td><td>NDC 58768-791-15</td><td>&amp;nbsp;clear lubricant eye gel&amp;nbsp;</td></tr><tr><td>Ketamine</td><td>Vedco Inc</td><td>NDC 50989-996-06</td><td></td></tr><tr><td>Xylazine</td><td>Lloyd Laboratories</td><td>NADA 139-236</td><td></td></tr><tr><td>Acepromazine</td><td>Vedco Inc</td><td>NDC 50989-160-11</td><td></td></tr><tr><td>32-G Needle</td><td>Steriject</td><td>PRE-32013</td><td></td></tr><tr><td>1-ml syringe</td><td>BD</td><td>309659</td><td></td></tr><tr><td>Indocyanine Green</td><td>Pfaltz &amp;amp; Bauer</td><td>I01250</td><td></td></tr></tbody></table>

detecting abnormalities in choroidal vasculature in a mouse model of age-related macular degeneration by time-course indocyanine green angiography

Indocyanine Green&#160;Angiography (or ICGA) is a technique performed by ophthalmologists to diagnose abnormalities of the choroidal and retinal vasculature of various eye diseases such as age-related macular degeneration (AMD). ICGA is especially useful to image the posterior choroidal vasculature of the eye due to its capability of penetrating through the pigmented layer with its infrared spectrum. ICGA time course can be divided into early, middle, and late phases. The three phases provide valuable information on the pathology of eye problems. Although time-course ICGA by intravenous (IV) injection is widely used in the clinic for the diagnosis and management of choroid problems, ICGA by intraperitoneal injection (IP) is commonly used in animal research. Here we demonstrated the technique to obtain high-resolution ICGA time-course images in mice by tail-vein injection and confocal scanning laser ophthalmoscopy. We used this technique to image the choroidal lesions in a mouse model of age-related macular degeneration. Although it is much easier to introduce ICG to the mouse vasculature by IP, our data indicate that it is difficult to obtain reproducible ICGA time course images by IP-ICGA. In contrast, ICGA via tail vein injection provides high quality ICGA time-course images comparable to human studies. In addition, we showed that ICGA performed on albino mice gives clearer pictures of choroidal vessels than that performed on pigmented mice. We suggest that time-course IV-ICGA should become a standard practice in AMD research based on animal models.

We performed ICGA time course in HTRA1 transgenic mice and control WT littermates, both of which are on the CD1 background. The albino CD1 background was selected to facilitate indocyanine green angiography (ICGA) imaging (see DISCUSSION). Some aneurism like dilations began to appear in the early phase in the HTRA1 mouse (Figure 2, a red arrow indicates the dilation at the tip of a vessel and a red circle indicates a cluster type polypoidal lesion). Choroidal vessels are clearly visible in both WT and HTRA1 mice during this early fill-in stage of the ICG dye. In the middle phase, the hyperfluorescent lesions in the early phase became clearer and more lesions started to appear while the choroidal vessels started to fade in the HTRA1 mouse (yellow circles indicate the appearance of more lesions). In the late phase, choroidal lesions of the HTRA1 mouse became more &#34;distinct&#34; as all the vessels faded away into the background. The optic nerve head was dark in both WT and HTRA1 mice (green arrows). The key features of the three phases are similar to the ICGA time course in human AMD patients (early phase, 0-3 min; middle phase, 5-15 min; late phase, 18-22 min)1.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51061/51061fig1highres.jpg" src="/files/ftp_upload/51061/51061fig1.jpg" /> 
Figure 1. Comparison of FA and ICGA in imaging mouse retinal and choroidal vasculature. WT CD1 mice were imaged by IV-FA and IV-ICGA using a Multi-Modality Imaging System. Retinal vessels can be seen in both FA and ICGA. Choroidal vessels can only be seen in ICGA. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51061/51061fig1highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/51061/51061fig2highres.jpg" src="/files/ftp_upload/51061/51061fig2.jpg" /> 
Figure 2. ICGA time course of HTRA1 transgenic mice by IV injection. A WT control and a HTRA1 transgenic mouse were imaged by ICGA (with tail vein injection). A red arrow indicates the dilation at the tip of a vessel (single polyp) and a red circle indicates a cluster type polypoidal lesion, which appeared in the early phase. Yellow circles indicate several lesions that appeared in the middle phase. Green arrows point to the dark optic nerve head in both WT and HTRA1 mice. Note that polypoidal lesions appear in the early phase of ICGA and become more distinct in the middle phase as in human studies21-24. Discrete dot lesions appear in the middle phase and become clear in the late phases (e.g. the biggest yellow circle indicating three dot lesions). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51061/51061fig2highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/51061/51061fig3highres.jpg" src="/files/ftp_upload/51061/51061fig3.jpg" /> 
Figure 3. ICGA time course of HTRA1 transgenic mice by IP injection. HTRA1 transgenic mice were imaged 5, 12, and 20 min after IP injection of ICG. The images of the two panel rows were taken from two different HTRA1 transgenic mice. Note that the choroidal vasculature is mostly invisible even 5 min after injection (12 min for the mouse in the lower panels). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51061/51061fig3highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 4" fo:content-width="5in" fo:src="/files/ftp_upload/51061/51061fig4highres.jpg" src="/files/ftp_upload/51061/51061fig4.jpg" /> 
Figure 4. ICGA time course of a pigmented (C57Bl6) and an albino (CD1) mouse by IV injection. Note the difference in the clarity of choroidal vasculature between the pigmented and the albino mice. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51061/51061fig4highres.jpg" target="_blank">Click here to view larger image</a>.

Watch this Scientific Journal Video about Detecting Abnormalities in Choroidal Vasculature in a Mouse Model of Age-related Macular Degeneration by Time-course Indocyanine Green Angiography at JoVE.com

Detecting Abnormalities in Choroidal Vasculature in a Mouse Model of Age-related Macular Degeneration by Time-course Indocyanine Green Angiography

Indocyanine Green Angiography (or ICGA) performed by tail vein injection provides high quality ICGA time course images to characterize abnormalities in mouse choroid.

Indocyanine Green&#160;Angiography (or ICGA) is a technique performed by ophthalmologists to diagnose abnormalities of the choroidal and retinal ...

detecting-abnormalities-choroidal-vasculature-mouse-model-age-related

Nanyang Technological University

Research

JoVE Journal

Medicine

13.6K Views.  University of Utah Health Sciences Center. Indocyanine Green Angiography (or ICGA) performed by tail vein injection provides high quality ICGA time course images to characterize abnormalities in mouse choroid.

Video: Detecting Abnormalities in Choroidal Vasculature in a Mouse Model of Age-related Macular Degeneration by Time-course Indocyanine Green Angiography

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with distinct functions and gene expression profiles. The roles of microglial activation in health, injury and disease remain incompletely understood due to their dynamic and complex regulation in response to changes in their microenvironment. Thus, it is critical to non-invasively monitor and analyze changes in microglial activation over time in the intact organism. In vivo studies of microglial activation have been delayed by technical limitations to tracking microglial behavior without altering the CNS environment. This has been particularly challenging during chronic neurodegeneration, where long-term changes must be tracked. The retina, a CNS organ amenable to non-invasive live imaging, offers a powerful system to visualize and characterize the dynamics of microglia activation during chronic disorders.
This protocol outlines methods for long-term, in vivo imaging of retinal microglia, using confocal ophthalmoscopy (cSLO) and CX3CR1GFP/+ reporter mice, to visualize microglia with cellular resolution. Also, we describe methods to quantify monthly changes in cell activation and density in large cell subsets (200-300 cells per retina). We confirm the use of somal area as a useful metric for live tracking of microglial activation in the retina by applying automated threshold-based morphometric analysis of in vivo images. We use these live image acquisition and analyses strategies to monitor the dynamic changes in microglial activation and microgliosis during early stages of retinal neurodegeneration in a mouse model of chronic glaucoma. This approach should be useful to investigate the contributions of microglia to neuronal and axonal decline in chronic CNS disorders that affect the retina and optic nerve.

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia activation and microgliosis are key responses to chronic neurodegeneration. Here, we present methods for in vivo, long-term visualization of retinal CX3CR1-GFP+ microglial cells by confocal ophthalmoscopy, and for threshold and morphometric analyses to identify and quantify their activation. We monitor microglial changes during early stages of age-related glaucoma.

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with ...

A Mouse Model for Laser-induced Choroidal Neovascularization

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) research. By administering targeted laser injury to the RPE and Bruch&#8217;s membrane, the procedure induces angiogenesis, modeling the hallmark pathology observed in neovascular AMD. 
First developed in non-human primates, the laser-induced CNV model has come to be implemented into many other species, the most recent of which being the mouse. Mouse experiments are advantageously more cost-effective, experiments can be executed on a much faster timeline, and they allow the use of various transgenic models. The miniature size of the mouse eye, however, poses a particular challenge when performing the procedure. Manipulation of the eye to visualize the retina requires practice of fine dexterity skills as well as simultaneous hand-eye-foot coordination to operate the laser. However, once mastered, the model can be applied to study many aspects of neovascular AMD such as molecular mechanisms, the effect of genetic manipulations, and drug treatment effects. 
The laser-induced CNV model, though useful, is not a perfect model of the disease. The wild-type mouse eye is otherwise healthy, and the chorio-retinal environment does not mimic the pathologic changes in human AMD. Furthermore, injury-induced angiogenesis does not reflect the same pathways as angiogenesis occurring in an age-related and chronic disease state as in AMD.
Despite its shortcomings, the laser-induced CNV model is one of the best methods currently available to study the debilitating pathology of neovascular AMD. Its implementation has led to a deeper understanding of the pathogenesis of AMD, as well as contributing to the development of many of the AMD therapies currently available.

Here, we present the mouse laser-induced choroidal neovascularization (CNV) protocol, an experimental model that re-creates the vascular hallmarks of neovascular age-related macular degeneration (AMD). Once mastered, it can reliably and effectively induce CNV as a model system to test various experimental measures.

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) ...

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this protocol, we aim to guide the reader not simply through the technical considerations of generating laser-induced lesions to trigger neovascular processes, but rather focus on the powerful information that can be obtained from multimodal longitudinal in vivo imaging throughout the follow-up period.
The laser-induced mouse CNV model was generated by a diode laser administration. Multimodal in vivo imaging techniques were used to monitor CNV induction, progression and regression. First, spectral domain optical coherence tomography (SD-OCT) was performed immediately after the lasering to verify a break of Bruch&#39;s membrane. Subsequent in vivo imaging using fluorescein angiography (FA) confirmed successful damage of Bruch&#39;s membrane from serial images acquired at the choroidal level. Longitudinal follow-up of CNV proliferation and regression on days 5, 10, and 14 after the lasering was performed using both SD-OCT and FA. Simple and reliable grading of leaky CNV leasions from FA images is&#160;presented. Automated segmentation for measurement of total retinal thickness, combined with manual caliber application for measurement of retinal thickness at CNV sites, allow unbiased evaluation of the presence of edema. Finally, histological verification of CNV is performed using isolectin GS-IB4 staining on choroidal flatmounts. The staining is thresholded, and the isolectin-positive area is calculated with ImageJ.
This protocol is especially useful in therapeutics studies requiring high-throughput-like screening of CNV pathology as it allows fast, multimodal, and reliable classification of CNV pathology and retinal edema. In addition, high resolution SD-OCT enables the recording of other pathological hallmarks, such as the accumulation of subretinal or intraretinal fluid. However, this method does not provide a possibility to automate CNV volume analysis from SD-OCT images, which has to be performed manually.

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Here, we present the usefulness of longitudinal in vivo imaging in the follow-up of morphological changes of laser-induced choroidal neovascularization in mice.

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this ...

Neuroscience

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the present protocol, mice expressing green fluorescent protein (gfp) under the promoter of Cx3cr1 (BALB/c-Cx3cr1gfp/gfp) were used to image microglia cells in vivo in the retina. Microglia are resident macrophages of the retina and have been implicated in several retinal diseases1,2,3,4,5,6. This protocol provides a detailed approach for generation of retinal B-scans, with SD-OCT, and imaging of microglia cell distribution in Cx3cr1gfp/gfp mice with SLO in vivo, using an ophthalmic imaging platform system. The protocol can be used in several reporter mouse lines. However, there are some limitations to the protocol presented here. First, both SLO and SD-OCT, when used in the high-resolution mode, collect data with high axial resolution but the lateral resolution is lower (3.5 &#181;m and 6 &#181;m, respectively). Moreover, the focus and saturation level in SLO is highly dependent on parameter selection and correct alignment of the eye. Additionally, using devices designed for human patients in mice is challenging due to the higher total optical power of the mouse eye compared to the human eye; this can lead to lateral magnification inaccuracies7, which are also dependent on the magnification by the mouse lens among others. However, despite that the axial scan position is dependent upon lateral magnification, the axial SD-OCT measurements are accurate8.

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

This protocol describes how high-resolution imaging techniques such as spectral domain optical coherence tomography and scanning laser ophthalmoscopy can be utilized in small rodents, using an ophthalmic imaging platform system, to obtain information on retinal thickness and microglial cell distribution, respectively.

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the ...

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To properly leverage this greater translatability, there is a need to devise reproducible methods of drawing quantitative data from these images. Optical coherence tomography (OCT) imaging can resolve retinal histology at micrometer resolution and reveal functional differences in vascular blood flow. Here, we delineate noninvasive vascular readouts that we use to characterize pathological damage post vascular insult in an optimized mouse model of retinal vein occlusion (RVO). These readouts include live imaging analysis of retinal morphology, disorganization of retinal inner layers (DRIL) measure of capillary ischemia, and fluorescein angiography measures of retinal edema and vascular density. These techniques correspond directly to those used to examine patients with retinal disease in the clinic. Standardizing these methods enables direct and reproducible comparison of animal models with clinical phenotypes of ophthalmic disease, increasing the translational power of vascular injury models.

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Here, we present three data analysis protocols for fluorescein angiography (FA) and optical coherence tomography (OCT) images in the study of Retinal Vein Occlusion (RVO).

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To ...

Application of Optical Coherence Tomography to a Mouse Model of Retinopathy

Optical coherence tomography (OCT) offers a noninvasive method for the diagnosis of retinopathy. The OCT machine can capture retinal crosssectional images from which the retinal thickness can be calculated. Although OCT is widely used in clinical practice, its application in basic research is not as prevalent, especially in small animals such as mice. Because of the small size of their eyeballs, it is challenging to conduct fundus imaging examinations in mice. Therefore, a specialized retinal imaging system is required to accommodate OCT imaging on small animals. This article demonstrates a small-animal-specific system for OCT examination procedures and a detailed method for image analysis. The results of retinal OCT examination of very-low-density lipoprotein receptor (Vldlr) knockout mice and C57BL/6J mice are presented. The OCT images of C57BL/6J mice showed retinal layers, while those of Vldlr knockout mice showed subretinal neovascularization and retinal thinning. In summary, OCT examination could facilitate the noninvasive detection and measurement of retinopathy in mouse models.

Here, we describe an in vivo imaging technique using optical coherence tomography to facilitate the diagnosis and quantitative measurement of retinopathy in mice.

Optical coherence tomography (OCT) offers a noninvasive method for the diagnosis of retinopathy. The OCT machine can capture retinal crosssectional images ...

Image-Guided Optical Coherence Tomography to Assess Structural Changes in Rodent Retinas

Ocular diseases, such as age-related macular degeneration, glaucoma, retinitis pigmentosa, and uveitis, are always accompanied by retinal structural changes. These diseases affecting the fundus always exhibit typical abnormalities in certain cell types in the retina, including photoreceptor cells, retinal ganglion cells, cells in the retinal blood vessels, and cells in the choroidal vascular cells. Noninvasive, highly efficient, and adaptable imaging techniques are required for both clinical practice and basic research. Image-guided optical coherence tomography (OCT) satisfies these requirements because it combines fundus photography and high-resolution OCT, providing an accurate diagnosis of tiny lesions as well as important changes in the retinal architecture. This study details the procedures of data collection and data analysis for image-guided OCT and demonstrates its application in rodent models of choroidal neovascularization (CNV), optic nerve crush (ONC), light-induced retinal degeneration, and experimental autoimmune uveitis (EAU). This technique helps researchers in the eye field to identify rodent retinal structural changes conveniently, reliably, and tractably.

The protocol presented here details the procedures of data collection and data analysis for image-guided optical coherence tomography (OCT) and demonstrates its application in multiple rodent models of ocular diseases.

Ocular diseases, such as age-related macular degeneration, glaucoma, retinitis pigmentosa, and uveitis, are always accompanied by retinal structural ...

Measuring Retinal Vessel Diameter from Mouse Fluorescent Angiography Images

It is important to study the development of retinal vasculature in retinopathies in which abnormal vessel growth can ultimately lead to vision loss. Mutations in the microphthalmia-associated transcription factor (Mitf) gene show hypopigmentation, microphthalmia, retinal degeneration, and in some cases, blindness. In vivo imaging of the mouse retina by noninvasive means is vital for eye research. However, given its small size, mouse fundus imaging is difficult and might require specialized tools, maintenance, and training. In this study, we have developed a unique software enabling analysis of the retinal vessel diameter in mice with an automated program written in MATLAB. Fundus photographs were obtained with a commercial fundus camera system following an intraperitoneal injection of a fluorescein salt solution. Images were altered to enhance contrast, and the MATLAB program permitted extracting the mean vascular diameter automatically at a predefined distance from the optic disk. The vascular changes were examined in wild-type mice and mice with various mutations in the Mitf gene by analyzing the retinal vessel diameter. The custom-written MATLAB program developed here is practical, easy to use, and allows researchers to analyze the mean diameter and mean total diameter, as well as the number of vessels from the mouse retinal vasculature, conveniently and reliably.

Mouse retinal vasculature is particularly interesting in understanding the mechanisms of vascular pattern formation. This protocol automatically measures the diameter of mouse retinal vessels from fluorescent angiography fundus images at a fixed distance from the optic disk.

It is important to study the development of retinal vasculature in retinopathies in which abnormal vessel growth can ultimately lead to vision loss. ...

Characterizing Different Lesion Types of Laser-Induced CNV in Mouse

Characterization of Vascular Morphology of Neovascular Age-Related Macular Degeneration by Indocyanine Green Angiography

Age-related macular degeneration (AMD) is a leading cause of blindness among older individuals, and its prevalence is rapidly increasing due to the aging population. Choroidal neovascularization (CNV) or wet AMD, which accounts for 10%-20% of all AMD cases, is responsible for an alarming 80%-90% of AMD-related blindness. Current anti-VEGF therapies show suboptimal responses in approximately 50% of patients. Resistance to anti-VEGF treatment in CNV patients is often associated with arteriolar CNV, while responders tend to have capillary CNV. While fluorescein angiography (FA) is commonly used to assess leakage patterns in wet AMD patients and animal models, it does not provide information about CNV vascular morphology (arteriolar CNV vs. capillary CNV). This protocol introduces the use of indocyanine green angiography (ICGA) to characterize lesion types in laser-induced CNV mouse models. This method is crucial for investigating the mechanisms and treatment strategies for anti-VEGF resistance in wet AMD. It is recommended to incorporate ICGA alongside FA for comprehensive assessment of both leakage and vascular features of CNV in mechanistic and therapeutic studies.

Author Spotlight: Overcoming Anti-VEGF Resistance Through Advanced Vascular Morphology Assessment in Choroidal Neovascularization

Currently, fluorescein angiography (FA) is the preferred method for identifying leakage patterns in animal models of choroidal neovascularization (CNV). However, FA does not provide information about vascular morphology. This protocol outlines the use of indocyanine green angiography (ICGA) to characterize different lesion types of laser-induced CNV in mouse models.

Age-related macular degeneration (AMD) is a leading cause of blindness among older individuals, and its prevalence is rapidly increasing due to the aging ...

An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell (EC) proliferation assays using human retinal microvascular endothelial cells (HRMECs) or isolated primary retinal ECs are widely used in vitro models to study retinal angiogenesis. However, isolating pure murine retinal endothelial cells is technically challenging and retinal ECs may have different proliferation responses than choroidal endothelial cells and different cell/cell interactions. A highly reproducible ex vivo choroidal sprouting assay as a model of choroidal microvascular proliferation was developed. This model includes the interaction between choroid vasculature (EC, macrophages, pericytes) and retinal pigment epithelium (RPE). Mouse RPE/choroid/scleral explants are isolated and incubated in growth-factor-reduced basal membrane extract (BME) (day 0). Medium is changed every other day and choroid sprouting is quantified at day 6. The images of individual choroid explant are taken with an inverted phase microscope and the sprouting area is quantified using a semi-automated macro plug-in to the ImageJ software developed in this lab. This reproducible ex vivo choroidal sprouting assay can be used to assess compounds for potential treatment and for microvascular disease research to assess pathways involved in choroidal micro vessel proliferation using wild type and genetically modified mouse tissue.

This protocol presents choroid sprouting assay, an ex vivo model of microvascular proliferation. This assay can be used to assess pathways involved in proliferating choroidal micro vessels and assess drug treatments using wild type and genetically modified mouse tissue.

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell ...

Detecting Abnormalities in Choroidal Vasculature in a Mouse Model of Age-related Macular Degeneration by Time-course Indocyanine Green Angiography

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Chapters in this video

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

A Mouse Model for Laser-induced Choroidal Neovascularization

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

In Vivo Imaging of Cx3cr1^gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Application of Optical Coherence Tomography to a Mouse Model of Retinopathy

Image-Guided Optical Coherence Tomography to Assess Structural Changes in Rodent Retinas

Measuring Retinal Vessel Diameter from Mouse Fluorescent Angiography Images

Characterization of Vascular Morphology of Neovascular Age-Related Macular Degeneration by Indocyanine Green Angiography

An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis